JP2019511525A - Pre-enzyme composition for cancer treatment - Google Patents

Abstract

  The present invention relates to compositions, methods, uses and kits for treating cancer. In particular, the present invention is a composition and method of treating cancer in a subject comprising administering a fixed amount of chymotrypsinogen, wherein, for example, the weight ratio of chymotrypsinogen: trypsinogen is greater than 1: 1 However, it relates to compositions and methods for treating cancer that is less than 4: 1.

Description

  The present application relates to compositions, methods, uses and kits for treating cancer.

This application claims priority from US Patent Application No. 62 / 321,370, the entire content of which is incorporated herein in its entirety.

  In recent years, the use of proteases in the treatment of cancer has been suggested. Initially, fresh pancreatic enzyme extracts were contemplated as potential cancer therapies and several good experiments were performed using the Jersen mouse sarcoma model. Tumor regression was observed following injection of the protease enzyme trypsin into mice. The results obtained are of great interest, and crude enzyme extracts prepared from sheep pancreas were used to treat human cancer patients to reduce tumor progression and prolong survival time .

  The use of proenzyme (an inactive precursor form of the enzyme) was used to try to overcome the challenges faced by oral administration of the enzyme, with varying results. A proenzyme mixture containing trypsinogen, a proenzyme form of the serine protease inhibitor trypsin, has been shown to be useful in the treatment of carcinomas and is believed to be selectively activated on the surface of tumor cells. The mechanism of action of trypsin is believed to occur as proteolysis of tumor cells. Compositions containing chymotrypsinogen and trypsinogen have been shown to be effective in assays for cancer, for example, pancreatic and colon cancer (US Pat. No. 5,677,859).

International Publication No. 2011/0174434 brochure

  However, there is a need to provide new or improved cancer treatments.

  Reference to any prior art herein is neither an acknowledgment nor a suggestion that this prior art forms part of common general knowledge in any jurisdiction, or such prior art is understood and related by those skilled in the art. Neither is it perceived or implied that it may be reasonably expected to be considered and / or combined with some of the other prior art.

  The present invention is a composition for treating cancer in a subject, comprising chymotrypsinogen and trypsinogen, wherein the weight ratio of chymotrypsinogen: trypsinogen is greater than 1: 1 but less than 4: 1 (ie, chymotrypsinogen: trypsinogen). The composition is provided, wherein the amount of trypsinogen is more than 1-fold but less than 4-fold as compared to trypsinogen. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1.

  The present invention is a pharmaceutical composition for treating cancer in a subject, comprising chymotrypsinogen, trypsinogen and a pharmaceutically acceptable diluent, excipient or carrier, wherein the weight ratio of chymotrypsinogen: trypsinogen is Provided are pharmaceutical compositions that are greater than 1: 1 but less than 4: 1. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1. In one embodiment, the only active anti-tumor components present in the composition are chymotrypsinogen and trypsinogen.

  The present invention is a pharmaceutical composition for treating cancer in a subject, comprising chymotrypsinogen and trypsinogen as active ingredients, further comprising a pharmaceutically acceptable diluent, excipient or carrier, which comprises: Provided is a pharmaceutical composition wherein the weight ratio of trypsinogen is greater than 1: 1 but less than 4: 1. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1. In one embodiment, the only active anti-tumor components present in the composition are chymotrypsinogen and trypsinogen.

  The present invention is a pharmaceutical composition for treating cancer in a subject, comprising chymotrypsinogen and trypsinogen as main ingredients, further comprising a pharmaceutically acceptable diluent, excipient or carrier, which comprises: Provided is a pharmaceutical composition wherein the weight ratio of trypsinogen is greater than 1: 1 but less than 4: 1. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1. In one embodiment, the only active anti-tumor components present in the composition are chymotrypsinogen and trypsinogen.

  The present invention is a pharmaceutical composition for treating pancreatic cancer in a subject, comprising chymotrypsinogen, trypsinogen and a pharmaceutically acceptable diluent, excipient or carrier, wherein the weight ratio of chymotrypsinogen: trypsinogen is Provided a pharmaceutical composition that is greater than 1: 1 but less than 4: 1. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1.

  The present invention is a pharmaceutical composition for treating ovarian cancer in a subject comprising chymotrypsinogen, trypsinogen and a pharmaceutically acceptable diluent, excipient or carrier, wherein the weight ratio of chymotrypsinogen: trypsinogen is Provided a pharmaceutical composition that is greater than 1: 1 but less than 4: 1. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1.

  The present invention is also a pharmaceutical composition for use in the treatment of cancer in a subject, comprising chymotrypsinogen, trypsinogen and a pharmaceutically acceptable diluent, excipient or carrier, and a weight ratio of chymotrypsinogen: trypsinogen Provides a pharmaceutical composition that is greater than 1: 1 but less than 4: 1. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1.

  The present invention is also a pharmaceutical composition comprising chymotrypsinogen, trypsinogen and a pharmaceutically acceptable diluent, excipient or carrier for use in the treatment of cancer in a subject, wherein the weight ratio of chymotrypsinogen: trypsinogen Provides a pharmaceutical composition that is greater than 1: 1 but less than 4: 1. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1.

  The invention is also a unit dose composition comprising chymotrypsinogen and trypsinogen, wherein the unit dose is administered in an amount of 6 mg or more of chymotrypsinogen, or any other amount described herein. Provided that the chymotrypsinogen: trypsinogen weight ratio is greater than 1: 1 but less than 4: 1. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1. Preferably, chymotrypsinogen is in an amount of 9 mg, 15 mg, 24 mg, 72 mg, 210 mg, 600 mg, 1200 mg or more.

  The invention is also a unit dose composition comprising chymotrypsinogen and trypsinogen, wherein the unit dose is for administering trypsinogen in an amount of 1 mg or more, or in any other amount described herein. And the weight ratio of chymotrypsinogen: trypsinogen is greater than 1: 1 but less than 4: 1. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1. Preferably, trypsinogen is in an amount of 1.5 mg, 3 mg, 3.5 mg, 12 mg, 36 mg, 90 mg, 180 mg or more than 360 mg.

  In any aspect of the invention, the composition comprises or consists of chymotrypsinogen and trypsinogen, and the amount of chymotrypsinogen is 1.5 mg / kg, 2 mg / kg, 3.5 mg / kg, 5 mg / kg, 15 mg / kg, 20 mg / kg, 40 mg / kg, 45 mg / kg, 135 mg / kg, 250 mg / kg or 500 mg / kg or more. The amount of chymotrypsinogen and trypsinogen can be greater than or equal to any mg / kg value described herein.

  In any aspect of the invention, the composition for treating cancer in humans comprises or consists of chymotrypsinogen and trypsinogen, and the amount of chymotrypsinogen is 0.1 mg / kg, 0.15 mg / kg, 0.25 mg / kg, 0.4 mg / kg, 1.2 mg / kg, 3.5 mg / kg, 10 mg / kg, 20 mg / kg or 40 mg / kg or more. The amount of chymotrypsinogen and trypsinogen can be greater than or equal to any mg / kg value described herein.

  In any aspect of the invention, the composition comprises or consists of chymotrypsinogen and trypsinogen, wherein the amount of trypsinogen is 0.25 mg / kg, 0.4 mg / kg, 0.6 mg / kg, 0.8 mg / Kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 5 mg / kg, 7 mg / kg, 8 mg / kg, 20 mg / kg, 40 mg / kg or 80 mg / kg or more. The amount of chymotrypsinogen and trypsinogen can be greater than or equal to any mg / kg value described herein.

  In any aspect of the invention, the composition for treating cancer in humans comprises or consists of chymotrypsinogen and trypsinogen, wherein the amount of trypsinogen is 0.02 mg / kg, 0.03 mg / kg, 0 .05 mg / kg, 0.06 mg / kg, 0.2 mg / kg, 0.6 mg / kg, 1.5 mg / kg, 3 mg / kg or 6 mg / kg or more. The amount of chymotrypsinogen and trypsinogen can be greater than or equal to any mg / kg value described herein.

  In any aspect of the invention, the pharmaceutical composition comprises, consists of or is adapted to administer chymotrypsinogen and trypsinogen, and the amount of chymotrypsinogen is 1.5 mg / kg, 2 mg / kg. kg, 3.5 mg / kg, 5 mg / kg, 15 mg / kg, 20 mg / kg, 40 mg / kg, 45 mg / kg, 135 mg / kg, 250 mg / kg or 500 mg / kg or more. The amount of chymotrypsinogen and trypsinogen can be greater than or equal to any mg / kg value described herein.

  In any aspect of the invention, the pharmaceutical composition for use in humans comprises, consists of or is adapted to administer chymotrypsinogen and trypsinogen, the amount of chymotrypsinogen being 0.1 mg / Kg, 0.15 mg / kg, 0.25 mg / kg, 0.4 mg / kg, 1.2 mg / kg, 3.5 mg / kg, 10 mg / kg, 20 mg / kg or 40 mg / kg or more. The amount of chymotrypsinogen may be at or above any mg / kg value described herein.

  In any aspect of the invention, the pharmaceutical composition comprises, consists of or is adapted to administer chymotrypsinogen and trypsinogen, and the amount of trypsinogen administered is 0.25 mg / kg, 0.4 mg / kg, 0.6 mg / kg, 0.8 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 5 mg / kg, 7 mg / kg, 8 mg / kg, 20 mg / kg, 40 mg / kg or 80 mg / kg or more. The amount of trypsinogen can be greater than or equal to any mg / kg value described herein.

  In any aspect of the invention, the pharmaceutical composition for use in humans comprises, consists of or is adapted to administer chymotrypsinogen and trypsinogen, and the amount of trypsinogen administered is 0 .02 mg / kg, 0.03 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.2 mg / kg, 0.6 mg / kg, 1.5 mg / kg, 3 mg / kg or 6 mg / kg or more is there. The amount of trypsinogen can be greater than or equal to any mg / kg value described herein.

  The present invention is also a method of treating cancer in a subject comprising administering chymotrypsinogen and trypsinogen, wherein the weight ratio of chymotrypsinogen: trypsinogen is greater than 1: 1 but less than 4: 1. , And thereby methods of treating cancer. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1.

  The present invention is a method of treating a solid tumor comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of trypsinogen and chymotrypsinogen, wherein the composition does not contain amylase, and chymotrypsinogen: The weight ratio of trypsinogen is greater than 1: 1 but less than 4: 1.

  The present invention is a method of treating a solid tumor comprising administering to the subject a pharmaceutical composition consisting essentially of a therapeutically effective amount of trypsinogen and chymotrypsinogen, wherein the weight ratio of chymotrypsinogen: trypsinogen is 1 Including methods that are greater than 1 but less than 4: 1.

The invention also relates to
A method of treating a solid tumor comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a vehicle or diluent; and a pre-protease composition consisting of trypsinogen and chymotrypsinogen, wherein the chymotrypsinogen comprises: trypsinogen Provides a method wherein the weight ratio of is greater than 1: 1 but less than 4: 1.

  The present invention is a method of treating cancer in a subject comprising administering chymotrypsinogen and trypsinogen, wherein chymotrypsinogen is administered in an amount of more than 0.1 mg / kg, and the weight ratio of chymotrypsinogen: trypsinogen is , Greater than 1: 1 but less than 4: 1, including methods of treating cancer. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1. Preferably, chymotrypsinogen is administered in an amount of more than 1 mg / kg.

  The present invention is a method of treating cancer in a subject comprising administering chymotrypsinogen and trypsinogen, wherein trypsinogen is administered in an amount of more than 0.02 mg / kg, and the weight ratio of chymotrypsinogen: trypsinogen is More than 1: 1 but less than 4: 1, including methods of treating cancer. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1. Preferably, chymotrypsinogen is administered in an amount of more than 0.2 mg / kg.

  Unless otherwise specified, as used herein, greater than 1: 1 but less than 4: 1 includes 3: 1 and 2: 1. In other words, the weight ratio of chymotrypsinogen: trypsinogen which is greater than 1: 1 but less than 4: 1 in any aspect of the invention includes 3: 1 or 2: 1 embodiments.

  The present invention includes methods of treating fibrosarcoma in a subject comprising administering chymotrypsinogen and trypsinogen, thereby treating fibrosarcoma in the subject. Preferably, amylase is not administered to the subject. The weight ratio of chymotrypsinogen: trypsinogen can be more than 1: 1, more than 2: 1, more than 4: 1, more than 6: 1 or more than 8: 1. The weight ratio of chymotrypsinogen: trypsinogen is more than 1: 1, but less than 8: 1, more than 1: 1, but less than 6: 1, more than 1: 1, but less than 4: 1, or 1 : More than 1, but may be 2: 1 or less. The weight ratio may be about 2: 1, about 4: 1, about 6: 1 or about 8: 1.

  The invention also provides the use of chymotrypsinogen and trypsinogen in the manufacture of a medicament for the treatment of fibrosarcoma. Preferably, the medicament does not contain amylase. The weight ratio of chymotrypsinogen: trypsinogen can be more than 1: 1, more than 2: 1, more than 4: 1, more than 6: 1 or more than 8: 1. The weight ratio of chymotrypsinogen: trypsinogen is more than 1: 1, but less than 8: 1, more than 1: 1, but less than 6: 1, more than 1: 1, but less than 4: 1, or 1 : More than 1, but may be 2: 1 or less. The weight ratio may be about 2: 1, about 4: 1, about 6: 1 or about 8: 1.

  In any of the methods of the invention, the amount of chymotrypsinogen administered in single or multiple doses over a period of 24 hours is greater than 0.1 mg / kg but less than 500 mg / kg. Preferably, chymotrypsinogen is administered in an amount of more than 1 mg / kg.

  In any of the methods of the invention, the amount of trypsinogen administered in single or multiple doses over a period of 24 hours is at least greater than 0.02 mg / kg but less than 90 mg / kg. Preferably, trypsinogen is administered in an amount of more than 0.2 mg / kg.

  In any of the methods of the invention, administration of the amounts of trypsinogen and chymotrypsinogen does not result in any clinically observable adverse events in the subject one week after administration, one day after administration, or preferably one hour after administration. Clinically observable adverse events can be weight loss, redness and behavioral changes at the injection site, or any one or more of any other events described herein.

  The invention is also the use of chymotrypsinogen and trypsinogen in the manufacture of a medicament for the treatment of cancer, wherein the weight ratio of chymotrypsinogen: trypsinogen is more than 1: 1 but less than 4: 1, Provides a use to treat cancer. Preferably, the weight ratio is greater than 1: 1 but less than or equal to 2: 1. Preferably, the weight ratio of chymotrypsinogen: trypsinogen is about 2: 1. Preferably, the medicament is adapted to administer chymotrypsinogen in an amount of more than 1 mg / kg. Preferably, the medicament is adapted to administer trypsinogen in an amount of more than 0.2 mg / kg, or in excess of any other amount described herein.

  The present invention is also a kit for treating cancer, comprising at least one dosage unit, wherein the dosage unit comprises chymotrypsinogen, trypsinogen and a pharmaceutically acceptable diluent, excipient or carrier, The dosage unit provides a kit adapted to administer chymotrypsinogen and trypsinogen in any amount or mg / kg value or more described herein.

  Optionally, the kit also includes instructions instructing the user to administer a dosage unit in an amount greater than 1 mg / kg of chymotrypsinogen, or any other amount described herein.

  Optionally, the kit also includes instructions instructing the user to administer a dosage unit in an amount greater than 0.2 mg / kg of trypsinogen, or any other amount described herein.

  The method and pharmaceutical composition of the present invention are cancer and metastatic carcinoma, such as pancreatic cancer, esophageal cancer, colon cancer, intestinal cancer, uterine cancer, prostate cancer, ovarian cancer, brain cancer, gastric cancer, breast cancer, liver cancer, kidney It is useful for the treatment of cancer, malignant melanoma, fibrosarcoma or lung cancer. Preferably, the cancer is pancreatic cancer, colon cancer or ovarian cancer. More preferably, the cancer is pancreatic cancer. Preferably, the brain tumor is glioblastoma.

  In any of the methods or uses of the present invention, the method or use further comprises the step of identifying a subject having or at risk of developing a cancer. Preferably, the cancer is any of those described herein.

  In any aspect of the invention, the composition does not contain amylase or the method or use does not administer amylase, ie the composition is amylase free and the method is amylase free composition Including administration.

  In any aspect, embodiment or form of the invention described herein, the amount of chymotrypsinogen administered is 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3.5 mg / kg, 5 mg / kg. kg, 15 mg / kg, 20 mg / kg, 40 mg / kg, 45 mg / kg, 135 mg / kg, 250 mg / kg or 500 mg / kg or more. The chymotrypsinogen administered can be at or above any mg / kg value described herein.

  In any aspect, embodiment or form of the invention described herein, the amount of trypsinogen administered is 0.2 mg / kg, 0.25 mg / kg, 0.4 mg / kg, 0.6 mg / kg 0.8 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 5 mg / kg, 7 mg / kg, 8 mg / kg, 20 mg / kg, 40 mg / kg or 80 mg / kg or more. The trypsinogen administered can be at or above any mg / kg value described herein.

  In any aspect, embodiment or form of the invention described herein, the amount of chymotrypsinogen administered to a human is 0.1 mg / kg, 0.15 mg / kg, 0.25 mg / kg, 0.1. It may be 4 mg / kg, 1.2 mg / kg, 3.5 mg / kg, 10 mg / kg, 20 mg / kg or 40 mg / kg or more. The amount of chymotrypsinogen may be at or above any mg / kg value described herein.

  In any aspect, embodiment or form of the invention described herein, the amount of trypsinogen administered to a human is 0.02 mg / kg, 0.03 mg / kg, 0.05 mg / kg, 0.06 mg / Kg, 0.2 mg / kg, 0.6 mg / kg, 1.5 mg / kg, 3 mg / kg or 6 mg / kg or more. The amount of trypsinogen can be greater than or equal to any mg / kg value described herein.

  Preferably, in any aspect, embodiment or form of the invention described herein, the amount of chymotrypsinogen administered is 1 mg / kg to 41 mg / kg, 1.5 mg / kg to 500 mg / kg, 2 mg / Kg to 250 mg / kg, 3.5 mg / kg to 135 mg / kg, 5 mg / kg or 15 mg / kg to 45 mg / kg. The chymotrypsinogen administered can be in the range between any two mg / kg values described herein.

  Preferably, in any aspect, embodiment or form of the invention described herein, the amount of trypsinogen administered is 0.2 mg / kg to 7 mg / kg, 0.25 mg / kg to 80 mg / kg, 0.4 mg / kg to 40 mg / kg, 0.6 mg / kg to 20 mg / kg, 0.8 mg / kg to 8 mg / kg, or 2.5 mg / kg to more than 8 mg / kg. The trypsinogen administered can be in the range between any two mg / kg values described herein.

  In any of the compositions of the invention described above, the composition can be adapted to administer the relevant mg or mg / kg of chymotrypsinogen and trypsinogen to the subject.

  The present invention also provides any of the above aspects of the invention that includes a value that is "about" the above stated value. For example, a further aspect of the invention is any of the above aspects wherein a reference to 500 mg / kg is about 500 mg / kg. This is contemplated for all aspects or embodiments of the invention and for all values.

  In any aspect of the invention, chymotrypsinogen and trypsinogen are administered intravenously, intraperitoneally, subcutaneously or intramuscularly.

  In any aspect of the invention, chymotrypsinogen trypsinogen can be administered simultaneously or sequentially. For sequential administration, chymotrypsinogen and then trypsinogen can be administered first, or trypsinogen and then chymotrypsinogen can be administered first.

  Unless the context requires otherwise, the term "comprise" and variants of that term as used herein, such as "comprises", "comprises" and "comprises" “Comprised” does not exclude further additives, components, integers or steps.

  Further aspects of the invention and further embodiments of the aspects described in the above paragraphs are given as examples and become apparent from the following detailed description with reference to the accompanying drawings.

Effect of the combination of trypsinogen and chymotrypsinogen on the growth of A2780 human ovarian tumor cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC _{50 of} trypsinogen (3.174 mg / ml). Coefficient of daily interaction (CDI) values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of ACHN human kidney tumor cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (2.984 mg / ml). CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of C8161.9 human melanoma cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 (3.917 mg / ml) of trypsinogen. CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the proliferation of DAOY human brain tumor cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (2.654 mg / ml). CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the proliferation of DU145 human prostate tumor cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 (3.843 mg / ml) of trypsinogen. CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of G-361 human melanoma cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (16.05 mg / ml). CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of HCT116 human colorectal tumor cells. A combination assay of trypsinogen and chymotrypsinogen was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 (16.43 mg / ml) of trypsinogen. CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of HCT-15 human colorectal tumor cells. A combination assay of trypsinogen and chymotrypsinogen was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (2.977 mg / ml). CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of Hep3B2.1-7 human hepatoma cells. A combination assay of trypsinogen and chymotrypsinogen was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (2.483 mg / ml). CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the proliferation of HT-1080 human fibrosarcoma cells. A combination assay of trypsinogen and chymotrypsinogen was performed at a ratio of 1: 1, 1: 2, 1: 4, 1: 6 and 1: 8 based on the previously determined IC50 (4.224 mg / ml) of trypsinogen. CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of HT-29 human colorectal tumor cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 (15.12 mg / ml) of trypsinogen. CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the proliferation of HuH-7 human hepatoma cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2, 1: 4, 1: 6 and 1: 8 based on the previously determined IC50 (3.934 mg / ml) of trypsinogen. CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of MCF-7 human breast tumor cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (16.57 mg / ml). CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the proliferation of MES-SA human uterine tumor cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (7.806 mg / ml). CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of NCI-H460 human lung tumor cells. A combination assay of trypsinogen and chymotrypsinogen was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (4.028 mg / ml). CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of PC-3 human prostate tumor cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 (17.46 mg / ml) of trypsinogen. CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the growth of SK-OV-3 human ovarian tumor cells. A combined trypsinogen and chymotrypsinogen assay was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (8.662 mg / ml). CDI values were calculated as described. Effect of the combination of trypsinogen and chymotrypsinogen on the proliferation of SNB-19 human brain tumor cells. A combination assay of trypsinogen and chymotrypsinogen was performed at a ratio of 1: 1, 1: 2 and 1: 4 based on the previously determined IC50 of trypsinogen (3.945 mg / ml). CDI values were calculated as described.

  The invention as disclosed and defined herein is understood to extend to all alternative combinations of two or more of the individual features listed in the text or figures or apparent therefrom. All of these different combinations constitute various alternative aspects of the invention.

  Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the invention does not limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

  One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. The invention as disclosed and defined herein is understood to extend to all alternative combinations of two or more of the individual features listed in the text or figures or apparent therefrom. All of these different combinations constitute various alternative aspects of the invention.

  All patents and publications mentioned herein are incorporated in their entirety by reference.

  For the purpose of interpreting the specification, terms used in the singular also include the plural and vice versa.

  In one aspect, the invention is based on the surprising finding that chymotrypsinogen and trypsinogen can be administered at a weight ratio of more than 1: 1 but less than 4: 1. Surprisingly, significant growth inhibition of a wide range of tumor cells is observed without requiring more than 4 times the weight of chymotrypsinogen relative to trypsinogen. Previous publications have suggested that a weight ratio of chymotrypsinogen and trypsinogen of 6: 1 is optimal, but the inventors now surprisingly have a significant inhibitory effect on cancer cell growth. It was identified to be achieved using chymotrypsinogen, which is present in relatively small amounts relative to trypsinogen.

  In another aspect, the invention is based on the finding that chymotrypsinogen and trypsinogen are effective against fibrosarcoma.

  The chymotrypsinogen (which may be abbreviated herein as "C") is a proenzyme form of the enzyme chymotrypsin which preferentially cleaves the protein at the following amino acids: tyrosine, tryptophan, phenylalanine and leucine. The chymotrypsin can be selected from chymotrypsin A, chymotrypsin B (for example, B1 and B2 forms), chymotrypsin C, alpha chymar loft, avazyme, chymar, chymotest, enzeon, It may be called or include quimar, quimotolase, alpha-chimar, alpha-chymotrypsin A, alpha-chymotrypsin. Chymotrypsin C can be formed from porcine chymotrypsinogen C, or from bovine subunit II of procarboxypeptidase A, and preferentially cleaves the protein at the following amino acids: tyrosine, tryptophan, phenylalanine, leucine, methionine, glutamine, and asparagine Do. The chymotrypsinogens include chymotrypsinogen B1 and chymotrypsinogen B2.

  Trypsinogen (which can be abbreviated as "T" herein) is a pre-enzyme form of trypsin that cleaves proteins preferentially at arginine and lysine. Trypsin may be α-trypsin, β-trypsin, cochnase, parenzyme, parenzymol (parenzymol), tryptar (tryptar), trypure (trypure), pseudotrypsin, tryptase, tripcerim, sperm receptor hydrolase β -May be referred to as trypsin, or they may be formed by cleavage of one peptide bond from trypsinogen. Further peptide bond cleavage produces alpha and other isoforms. Multiple cationic and anionic trypsins can be isolated from the pancreas of many vertebrates and from lower species, such as crayfish, insects (cochnase) and microorganisms (Streptomyces griseus). In the normal process during digestion, inactive trypsinogen is activated by the enteropeptidase present in the intestinal mucosa, is a serine protease, and then cleaves the peptide bond on the carboxyl side of the basic amino acid / protein The acting enzyme forms trypsin.

  Trypsinogen and chymotrypsinogen used in any aspect of the present invention may be isolated, purified, substantially purified recombinant or synthetic.

  The preenzymes trypsinogen and chymotrypsinogen are selected from trypsin from chymotrypsin class 3.4.21.1 or 3.4.21.2 or class 3.4.21.4, or any other suitable resource ( It may be a precursor of an enzyme selected from the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology according to the classification of the International Union of Biochemistry and Molecular Biology. These enzymes are commercially available and may be of human, bovine or porcine origin.

The present invention relates to human weights of 50, 60, 70, 80, 90, 100 kg or more and 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.. Includes human conversion for all amounts of mg / kg mentioned herein based on body surface area of 8 m ² or more. Specifically, the amount per kg for human administration, and the method of calculation are mentioned in Example 5.

  As mentioned, the pre-enzyme form essentially provides an inactivated form of the enzyme which becomes active in situ (eg in vivo or in vitro activation). For example, activation of proenzyme (conversion of proenzyme to active enzyme) can occur upon contact with the surface of a tumor cell. The preenzymes trypsinogen and chymotrypsinogen are thought to be selectively activated to the enzymes trypsin and chymotrypsin when in contact with tumor cells and when not in contact with healthy cells. The use of proenzymes reduces the problems associated with providing active enzymes in situ, such as undesired reactions prior to reaching the target tumor cell target or inactivation of the enzyme.

  In the context of tumor cells, protease enzymes can act to degrade the cell wall of malignant cells by cleaving the amide bond present in the peptide chain of the cell wall (proteolysis). It is also understood that protease inhibitors which are present in non-malignant cells and which inhibit or reduce the effect of the enzyme on cell wall degradation are absent in malignant tumor cells. In addition to providing proteolytic activity, proprotease can upregulate the expression of β-catenin and E-cadherin in tumor cells. Intercellular adhesion is promoted by complex formation or binding between β-catenin and E-cadherin on the cell surface, and thus, increased expression of β-catenin and E-cadherin leads to an improvement in intercellular adhesion, thereby It results in the reduction of metastasis of tumor cells. Protease proenzymes may also provide other cellular activities, such as increased immune recognition or differentiation.

  A serious adverse event, experience or response is any adverse medical event that results in death at any dose and is life threatening (subject bears immediate death risk), subject / subject hospitalization or length of stay Long term or serious disability, resulting in incapacitation, or birth defects / birth defects.

  A clinically observable adverse event is any unwanted medical event in a subject who has been administered a pharmaceutical product or a clinical trial subject. Clinically observable adverse events may include any one of the events or observations described herein, particularly in the Examples. Typically, the period of observation is about one week after administration, about one day after administration, or preferably about one hour after administration. The duration of observation may be the time between administration of doses.

  "Treating" or "treatment" refers both to therapeutic treatment and prophylactic or preventative measures whose purpose is to prevent, ameliorate, reduce or slow down (attenuate) cancer or its spread (metastasis).

  In any of the methods of the invention, one or more of the following effects: to reduce recurrence of malignancy, to reduce metastasis of malignancy, to reduce the number or size of tumors, to promote differentiation of tumor cells, adhesion between cells It is possible to observe the expression of β-catenin and E-cadherin in malignancies, and the reduction of metastasis, the reduction of tumor cell ability to prevent immune recognition.

  "Preventing", "prevention", "preventive" or "preventive" refers to a condition, disease, disorder, or phenotype, such as, for example, avoiding or preventing the occurrence of an abnormality or condition, protection therefrom, Or refers to the protection of its occurrence. Subjects in need of prevention may be prone to develop a condition.

  The terms “ameliorate” or “improvement” refer to the reduction, reduction or elimination of a condition, disease, disorder or phenotype, eg, an abnormality or condition. The subject in need of treatment may already have the condition or be prone to have the condition or may be one in which the condition is to be prevented.

  "Subject" includes mammals. The mammal may be human or may be a domestic animal, a zoo animal, or a companion animal. In particular, while the methods of the invention are suitable for human medical treatment, they are suitable for veterinary treatment, for example companion animals such as dogs and cats, and livestock such as horses, cattle and sheep, or It is specifically contemplated that it is also applicable to the treatment of zoo animals such as felines, canines, bovines and ungulates. The subject may suffer from cancer or other disorder, and may not suffer from cancer or other disorder (ie, has no detectable disease).

  The typical weight of a human subject may be 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 or more than 110 kg.

  The term "therapeutically effective amount" refers to the amount of a drug or compound in a composition or composition capable of treating, preventing or ameliorating cancer or its spread (metastasis). A therapeutically effective amount can be determined experimentally and routinely for the treatment of cancer, resulting in an increase in life expectancy.

  As used herein, "adapted for administration" refers to a composition having the ability to administer trypsinogen and chymotrypsinogen in a single dose or multiple doses at a defined amount or concentration.

  The methods of the invention described herein include the treatment of neoplasia and related disease states, cancer, tumors, malignant and metastatic disease states. Tissues and organs associated with solid tumors and metastases that can be treated by the methods or pharmaceutical compositions of the present invention include, but are not limited to, bile duct, bladder, blood, brain, breast, cervix, colon , Endometrium, esophagus, head, neck, kidney, pharynx, liver, lung, medulla, melanin, ovary, pancreas, prostate, rectum, kidney, retina, skin, stomach, testis, thyroid, urinary tract and uterus Can be mentioned.

  The methods and pharmaceutical compositions of the present invention are for the treatment of the following types of cancer and metastatic carcinoma: pancreatic cancer, esophageal cancer, colon cancer, intestinal cancer, prostate cancer, ovarian cancer, gastric cancer, breast cancer, malignant melanoma or lung cancer It is useful. Preferably, the cancer is pancreatic cancer, colon cancer or ovarian cancer. More preferably, the cancer is pancreatic cancer. The metastatic potential of a carcinoma can be low, moderate or high.

  The methods and pharmaceutical compositions of the present invention may provide, for example, a multi-effect approach to treating cancer by increasing tumor cell apoptosis, cell adhesion, differentiation and immunogenicity (targeting and removal by the immune system). . Thus, it is beneficial to perform the treatment in the absence of any other treatment that may suppress or damage the immune system.

  The methods or uses of the present invention can be supplemented with other conventional anti-cancer therapeutic approaches directed to the treatment or prevention of proliferative disorders (eg, tumors). For example, such methods can be used in preventative cancer prevention, prevention of postoperative cancer recurrence and metastasis, and as an adjuvant for other conventional cancer therapies.

  The pharmaceutical compositions of the present invention may be, for example, conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the desired mode of administration (eg, excipients, binders, preservatives, stabilizers, dressings Perfumes and the like) can be formulated by use according to the art, eg, those well known in the art of pharmaceutical formulation.

  The pharmaceutical composition of the invention may be administered by any suitable means, for example orally, for example in the form of tablets, capsules, granules or powders; sublingually; buccally; parenterally For example, by subcutaneous, intravenous, intramuscular, or intracapsular injection or infusion techniques (eg, as a sterile injectable water or non-aqueous solution or suspension); nasally, eg, by inhalation spray; topically Or in the form of a cream or ointment; or for rectal administration, eg in the form of a suppository; in a dosage unit formulation containing a non-toxic pharmaceutically acceptable vehicle or diluent . These can be administered, for example, in a form suitable for immediate release or sustained release, for example, through the use of a device, such as a subcutaneous implant, an encapsulated spheroid or an osmotic pump.

  In addition to primates, such as humans, various other mammals can be treated according to the method of the tenth aspect. For example, mammals such as, but not limited to, cattle, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, The rodent or murine species can be treated.

  The term "pharmaceutically acceptable" as used herein means a carrier, diluent or excipient which is not harmful to the recipient.

  The terms “administration” and / or “administering” should be understood to mean providing an individual in need of treatment.

  The individual in need of treatment may be an individual who has been diagnosed with or at risk of developing any one of the cancers described herein.

  The pharmaceutical composition of the invention, and the formulations or formulations thereof, can be prepared by mixing together the components of the composition, ie chymotrypsinogen and trypsinogen. The mixing can be carried out continuously or simultaneously.

  The pharmaceutical compositions of the invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active agent and proprotease into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active agent and the proprotease with the liquid carrier and / or the finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation Ru. The active agent and proprotease are provided in a unit dosage form in sufficient amounts to produce the desired effect on the disease process or condition after single or repeated administration.

  The pharmaceutical composition of the present invention may be used orally, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. In a suitable form. Compositions intended for oral use can be prepared according to any method known in the art of the manufacture of pharmaceutical compositions, such compositions providing pharmaceutically elegant and palatable preparations May contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservatives. The tablet contains the proprotease and active agent of the first and second aspects in a mixture with non-toxic pharmaceutically acceptable excipients suitable for tablet manufacture. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granules and disintegrants, such as corn starch or alginic acid; binders, such as starch, gelatin Or acacia, and lubricants, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl diastearate can be used. These may be coated to form an osmotic therapeutic tablet for controlled release.

  Formulations for oral use are as hard gelatine capsules in which the proprotease and active agent of the first and second aspects are mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or It may also be provided as a soft gelatin capsule wherein the proprotease and active agent of the first and second aspects are mixed with water or an oily medium, such as peanut oil, liquid paraffin or olive oil.

  The aqueous suspension contains the active agent and the protease proenzyme in a mixture with excipients suitable for the preparation of the aqueous suspension. Such excipients are, for example, suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, n-methyl-pyrrolidone, gum tragacanth and gum acacia, dispersing or wetting. The agent is a naturally occurring phosphatide, such as lecithin, or a condensation product of an alkylene oxide and a fatty acid, such as polyoxyethylene stearate, or a condensation product of ethylene oxide and a long chain aliphatic alcohol, such as heptadecaethyleneoxy Condensation products of cetanol or ethylene oxide with a partial ester derived from fatty acid and hexitol, such as polyoxyethylene sorbitol monooleate or ethylene oxide, Condensation products of ethylene oxide with partial esters derived from fatty acid and a hexitol anhydride, for example polyethylene sorbitan monooleate. The aqueous suspension comprises one or more preservatives, such as ethyl or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, For example, it may also contain sucrose or saccharin.

  Oily suspensions may be formulated by suspending the active agent and proprotease in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those described above, and flavoring agents may be added to provide a palatable oral preparation. These can be preserved by the addition of an anti-oxidant such as ascorbic acid.

  Dispersible powders and granules suitable for the preparation of an aqueous suspension by the addition of water comprise a pre-protease and an active agent according to the first and second aspects as a dispersing or wetting agent, a suspending agent and one Provided in a mixture with the above preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

  Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or mixtures thereof. Suitable emulsifiers are naturally occurring gums such as acacia gum or tragacanth gum, naturally occurring phosphatides such as soy, lecithin, and esters or partial esters derived from fatty acids and anhydrous hexitols such as sorbitan monooleate, and said partial esters And the condensation product of ethylene oxide with, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

  Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol and sucrose. They may also contain demulcents, preservatives and flavors and colors.

  The pharmaceutical compositions of the invention may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The pharmaceutical composition of the first and second aspects may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol It may be Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

  In a specific embodiment, the pharmaceutical composition of the invention is formulated as a suppository for rectal administration of the drug. These formulations are suitable, which are solid at ambient temperature, but liquid at rectal temperature, so that the proprotease and active agent of the first and second aspects are liquid at the rectum temperature and thus melt in the rectum to release the drug. It can be prepared by mixing with a non-irritating excipient. Such materials include cocoa butter and polyethylene glycols. Rectal administration can be used to eliminate the enteric liver first pass effect in the gastrointestinal tract associated with oral administration of the enzyme.

  The pharmaceutical composition of the present invention can also be formulated in liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. Liposomal formulations may contain stabilizers, preservatives, excipients, and the like. Preferred lipids are phospholipids and phosphatidyl choline, both natural and synthetic. Methods to form liposomes are known in the art.

  The pharmaceutical compositions of the invention can be included in a container, pack, or dispenser together with instructions for administration. Containers, packs to be taken separately or together at the same time or different times in the use or method of the invention as described herein, wherein the proprotease and the active agent of the pharmaceutical composition and optionally the additional active agent Or can be provided as discrete components in the dispenser.

  The invention as disclosed and defined herein is understood to extend to all alternative combinations of two or more of the individual features listed in the text or figures or apparent therefrom. All of these different combinations constitute various alternative aspects of the invention.

Example 1

Cell culture The following table shows the growth conditions and initial cell seeding density of cells per well used in all IC ₅₀ measurements and combination assays. Cells were cultured at 37 ° C. in a humidified cell culture incubator supplemented with 95% air / 5% CO ₂ .

  All cell lines below were sourced from American Type Culture Collection (ATCC) (Rockville, MD, USA): 786-0, ACHN, BT-474, DAOY, DU145, G-361, HCT116, HCT-15, Hep3B2.1-7, HL-60, HT-1080, HT-29, MCF-7, MDA-MB-231, MES-SA, NCI-H460, NCI-H82, PC-3, SK-OV-3, U-87 MG.

  A2780 cell line was obtained from the National Cancer Institute (NCI) (Bethesda, MD, USA).

  The C8161.9 cell line was obtained from the Gavin Robertson PhD laboratory (College of Medicine, Pennsylvania State University, Hershey, PA, USA).

  HuH-7 cell lines were obtained from the Japanese Collection of Research Bioresources (JCRB) cell bank (Osaka, Japan).

  SNB-19 and Raji cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (German Collection of Microorganisms and Cell Cultures) (Braunschweig, Germany).

  All cell lines were utilized in the assay up to passage 10.

examined goods

Test Article Formulations Trypsinogen and chymotrypsinogen were dissolved directly in appropriate cell culture medium and added immediately to the cells.

Cell Proliferation Assay Cell lines correspond to multiple tumor types and include: DAOY, SNB-19 and U87-MG for brain tumors; BT-474, MDA-MB-231 and MCF-7 for breast tumors; HCT116, HCT-15 and HT-29 for colon tumors; HT-1080 for fibrosarcoma; ACHN and 786-O for kidney tumors; HL-60 and Raji for leukemia; Hep3B2.1-7 and HuH-7 for liver tumors; NCI-H460 and NCI-H82 for lung tumors; C8161.9 and G-361 for melanomas; PC-3 and DU145 for prostate tumors; A2780 and SK-OV-3 for ovarian tumors; and MES-SA for uterine tumors.

  The test article was added to the cells 24 hours after seeding for combination assay. Test article concentrations were tested in triplicate for each cell line. Seventy-two hours after addition of the test article, the CellTiter-Blue® assay was performed on all plates.

The concentration of trypsinogen used in the combination assay was based on the IC ₅₀ calculated from a single test article experiment. Depending on the concentration of trypsinogen for each individual cell line, the concentration of chymotrypsinogen was determined in the following ratios: 1: 1, 1: 2, 1: 4, 1: 6 and 1: 8 (trypsinogen: chymotrypsinogen) . Controls consisted of growth medium alone and untreated cells and growth medium (untreated control).

The assay controls used were a phosphate buffered saline control as vehicle and growth media control as vehicle for Triton-x100 as positive control and growth media only (background). Background subtraction was not performed for the determination of IC ₅₀ values.

CellTiter-Blue (R) Assay After incubation of cells in medium containing test article, 10 [mu] L of CellTiter-Blue (R) was added to each well and then incubated with cells for up to 6 hours. The fluorescence was measured using a Spectramax Gemini XPS fluorometer (560 nm excitation, 590 nm fluorescence). All data were recorded and entered into a Microsoft® Excel spreadsheet for interpretation.

Calculations Data collected from the CellTiter-Blue® assay were plotted as dose response curves for IC ₅₀ determination. Relative fluorescence units (RFU) were plotted against compound concentration. In these plots, the X-axis (compound concentration) was expressed on a logarithmic scale. IC ₅₀ concentrations are half (50%) of the maximal inhibitory concentration (IC) for each compound via the variable slope curve fitting algorithm using GraphPad Prism version 6.0eMac OSX version (GraphPad Software, San Diego California, USA) Calculated as).

For combination studies, the drug interaction coefficient (CDI) is given by the following equation:
Where TC is the growth inhibition of the combination of trypsinogen and chymotrypsinogen, T is the growth inhibition of the single agent trypsinogen, and C is the growth inhibition of the single agent chymotrypsinogen. CDI values less than 1 indicate drug synergy of trypsinogen and chymotrypsinogen, while values above 1 indicate their antagonistic interactions.

Example 2
Cell growth inhibition by trypsinogen and chymotrypsinogen in human cancer Treatment with the combination of chymotrypsinogen (C) and trypsinogen (T) is more than 1: 1 based on CDI for the cell lines described in Figures 1-18 and Tables 1-18. And demonstrated greater growth inhibition as compared to 1: 1 at a ratio of less than 4: 1. The CDI values for each combination in all cell lines tested are shown in the figures and tables.

  In addition, the growth inhibition of liver tumor cells observed for ratios greater than 1: 1 but less than 4: 1 was equal to ratios of 4: 1, 6: 1 and 8: 1.

  Furthermore, growth inhibition of fibrosarcoma cells was significant at all ratios tested, including 2: 1, 4: 1, 6: 1 and 8: 1. The chymotrypsinogen and trypsinogen showed a significant synergistic effect on growth inhibition at all ratios tested above 1: 1, ie 2: 1, 4: 1, 6: 1 and 8: 1.

  Tables 1-18 below show the mean fluorescence of three replicates (relative fluorescence units-RFU), standard error of the sample mean (SEM), growth inhibition (1 = no growth inhibition, less than 1 = growth inhibition) and CDI (drug Interaction factor CDI values less than -1 indicate drug synergy of trypsinogen and chymotrypsinogen, while values greater than 1 indicate their antagonistic interaction).

  The invention as disclosed and defined herein is understood to extend to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims (26)

  A composition for treating cancer in a subject comprising chymotrypsinogen and trypsinogen, wherein the weight ratio of chymotrypsinogen: trypsinogen is greater than 1: 1 but less than 4: 1.   The composition of claim 1 wherein said weight ratio is greater than 1: 1 but less than or equal to 2: 1.   The composition according to claim 1 or 2, wherein the weight ratio is 2: 1.   The composition according to claim 1, wherein the weight ratio is 3: 1.   The composition according to any one of claims 1 to 4, which does not contain amylase.   A method of treating cancer in a subject comprising administering chymotrypsinogen and trypsinogen, wherein the weight ratio of chymotrypsinogen: trypsinogen is greater than 1: 1 but less than 4: 1, whereby said subject To treat cancer in the   7. The method of claim 6, wherein the weight ratio is greater than 1: 1 but less than 2: 1.   The method according to claim 6 or 7, wherein the weight ratio is 2: 1.   The method according to claim 6 or 7, wherein the weight ratio is 3: 1.   The method according to any one of claims 6 to 9, which does not include administration of amylase.   Use of chymotrypsinogen and trypsinogen in the manufacture of a medicament for the treatment of cancer wherein the weight ratio of chymotrypsinogen: trypsinogen is greater than 1: 1 but less than 4: 1.   The use according to claim 11, wherein the weight ratio is more than 1: 1 but less than 2: 1.   The use according to claim 11 or 12, wherein the weight ratio is 2: 1.   The use according to claim 11 or 12, wherein the weight ratio is 3: 1.   The use according to any one of claims 11 to 14, wherein said method does not comprise the administration of amylase.   The cancer according to any one of claims 6 to 15, wherein the cancer is any one of pancreatic cancer, esophageal cancer, colon cancer, intestinal cancer, prostate cancer, ovarian cancer, gastric cancer, breast cancer, liver cancer, malignant melanoma or lung cancer. The method or use according to one of the items.   The method or use according to any one of claims 6 to 15, wherein the cancer is ovarian cancer, melanoma, brain tumor, prostate cancer, colorectal cancer, liver cancer or lung cancer.   The method or use according to any one of claims 6 to 15, wherein the cancer is pancreatic cancer, colon cancer or ovarian cancer.   The method or use according to any one of claims 6 to 15, wherein the cancer is a pancreatic cancer.   A method of treating fibrosarcoma in a subject, comprising administering chymotrypsinogen and trypsinogen, thereby treating fibrosarcoma in the subject.   21. The method of claim 20, wherein the weight ratio of chymotrypsinogen: trypsinogen is greater than 1: 1 but not more than 8: 1.   22. The method of claim 21, wherein the weight ratio is greater than 1: 1 but less than or equal to 6: 1.   22. The method of claim 21, wherein the weight ratio is greater than 1: 1 but less than or equal to 4: 1.   22. The method of claim 21, wherein the weight ratio is greater than 1: 1 but less than or equal to 2: 1.   25. The method of any of claims 20-24, wherein the weight ratio is about 2: 1, about 4: 1, about 6: 1 or about 8: 1.   26. The method of any one of claims 20-25, which does not include administration of amylase.